Clear cell renal cell carcinoma (ccRCC) is the most common form of kidney malignancy. It is both chemotherapy and radiation resistant and current treatment options for this disease are limited, particularly because 1 out of 4 patients are initialy diagnosed at a disseminated, metastatic disease state. Underlying these clinical characteristics are a number of changes at the molecular level, the most pronounced of which are metabolic alterations that promote malignancy. One of these modifications is to the urea cycle, which is the second most underexpressed metabolic pathway in ccRCC. The urea cycle occurs in the kidney and is involved in detoxification of free ammonia species. Two key enzymes in this cycle are arginase II (ARG2) and argininosuccinate synthase 1 (ASS1); ARG2 is included on chromosome 14q and loss of this region correlates with high malignancy grade and a poorer prognosis in ccRCC. Data from nearly 500 primary ccRCC tumors in The Cancer Genome Atlas (TCGA) dataset demonstrates that ARG2 exhibits copy number loss in 40% of ccRCC and is underexpressed at the mRNA level, whereas ASS1 is significantly underexpressed, but not deleted, in ccRCC compared to normal kidney tissue. In preliminary experiments, we have demonstrated that re-expressing these enzymes in ccRCC cell lines suppresses proliferation in vitro and in vivo during xenograft tumor growth. Furthermore, re-expression of ARG2 and ASS1 reduces glycolytic flux and decreases mTOR activity. The central hypothesis of this proposal is that ARG2 and ASS1 act as tumor suppressors by modulating urea cycle and glycolytic metabolism and by repressing mTORC1 dependent growth pathways. Based on this hypothesis, the following specific aims will be pursued: Specific Aim 1: To determine the role ARG2 and ASS1 play in mediating mTORC1 driven proliferation in ccRCC. Specific Aim 2: To determine how metabolic alterations caused by ARG2 and ASS1 re-expression influence the pathogenesis of ccRCC. Completion of these studies will require a combination of in vitro and in vivo methods that include biochemistry, cell biology, immunohistochemistry, and metabolic flux analysis. The objective of this project is to determine the molecular mechanisms by which ARG2 and ASS1 loss alters cellular metabolism to promote ccRCC tumor growth. Delineating these mechanisms will provide new therapeutic avenues to target in this disease.